The present invention relates to an immunologically active coated solid phase for solid phase immunological assays in particular for solid phase radioimmuno assay techniques, to methods for preparing immunologically active solid phase and to immunological test methods and kits using same.
It is well known in the medical art that a variety of chemical substances, either as such or in form of a complex with a body protein, have antigenic activity, that is they are capable of acting as an antigen; i.e., when introduced into animals they are capable of eliciting an immune response and causing the formation of a specific antibody or immunological binder.
In the following specification and claims the term "antigen" designates all substances which, as such, or in form of a protein-complex, have antigenic activity. Antigens include high molecular substances such as proteins and other polypeptides and polysaccharides, for example many proteinaceous components of human body liquids, hormones, bacteria-produced toxins, virus, etc., lower molecular weight chemicals including many drugs which are so-called haptens, that is they react with a body protein to form a protein-complex with antigenic activity, as well as lower weight substances, which as such are capable of antigenic activity.
Antibodies are proteins which exhibit a specific immunological activity against the antigen which caused their formation. Antibodies have a molecular weight in the range of 150,000 and are very similar in their overall protein-structure, but are distinguished from each other by their specific affinity to different antigens.
Certain low molecular weight antigens are not capable of causing formation of an antibody, but elicit the formation of a lower molecular weight peptide, e.g. a peptide having a molecular weight in the range of between about 5,000 and 120,000 which possesses an antibody-like specific immunological activity against the antigen which caused their formation, that is, they are capable of selectively binding these antigens in an immunological reaction. These peptides are known as immunological binders.
Immunological methods for inducing antibody formation or formation of an immunological binder in animals and recovering the antibody or immunological binder from the animal, e.g., in form of an antiserum are well known in the art.
In the following specification and claims, the term "antibody" is meant to denote a proteinaceous material which exhibits antibody activity, that is, the antibody in substantially pure form or in form of a mixture having a high antibody-activity, e.g., an antiserum. The term "immunologically active binder" is meant to denote a peptide material which exhibits antibody-like immunological binding activity, that is, the peptide is substantially pure form or in form of mixture having a high antibody-like binding activity, e.g., an antiserum.
In the following specification and claims, the term "immunologically active agent" is meant to include antigens, antibodies and immunologically active binders.
Immunological diagnostic test methods generally comprise reacting a liquid in which a given antigen is to be determined with a reagent containing a known amount of an antibody or immunological binder, or reacting a reagent containing a known amount of antigen with a liquid wherein an antibody or immunological binder is to be determined, separating the antigen/antibody or antigen/immunological binder complex from the unreacted components and determining the residual amount of unreacted antigen or antibody, or the amount of reacted antigen or antibody in the complex reaction product.
Different methods which are known in the art can be used for the determination including microscopic and electromicroscopic examination, fluorescent method wherein a dyestuff is used to make the antigen or the antibody visible and radioimmuno assay (in the following, abbreviated as "RIA") techniques.
In radioimmunological methods, a known amount of e.g. an antigen, which is labeled with a radioactive isotope, is added to the liquid, the antigen of which is to be determined. The amounts of labeled and unlabeled antigen which react with the antibody are in proportion to their relative concentrations in the test liquid. The radioactivity of the antigen/antibody reaction product or of the unreacted residual antigen is measured.
For calculation of the antigen content of the original test liquid, the measured amount of radioactivity is compared with a standard curve which is prepared by reacting the same antibody reagent with standard samples containing known amounts of labeled and unlabeled antigen.
For any quantitative immunological test procedures, it is mandatory that the content of immunologically active agent in the reagent which is used in a test series is the same throughout the test series, and that quantitative separation between the complex reaction product and unreacted material can be achieved.
For facilitating the separation of the complex reaction product from the reaction mixture, solid phase immuno assay techniques have been developed for immunological tests involving antigen/antibody reactions. In these solid phase immuno assays, the antibody-containing reagent is a solid organic polymer substrate onto the surface of which the antibody is bonded.
Polymeric organic materials possess a certain adsorption capacity for adsorbing proteinaceous substances on their surfaces. The degree of adsorption and affinity of the polymer towards the adsorbed substance is dependent on the molecular weight of the adsorbed substance and decreases with decreasing molecular weight of the latter.
The technique of solid phase RIA was introduced by Catt and coworkers. The bonded antibody, e.g., an antiserum, will selectively react with, and bind antigen for which it is specific. When radioactively labeled antigen is added to the sample to be assayed, such as blood serum, urine, etc., labeled and unlabeled antigen will be bound in proportion to their concentration in the test sample. Thus, by incubating the antibody containing solid phase with the test sample, and then counting the radiation content of, e.g., the solid phase, the original concentration of unlabeled antigen therein can be determined.
Of course, the amount of antigen which is bonded to the solid phase during the assay will depend on the amount of antibody which is present on the surface of the solid phase. Therefore, the accuracy of solid phase immuno assays, in particular, solid phase radioimmuno assays and thus their practical usefulness, will depend on the availability of a method by means of which uniform and reproducible antibody-coated solid phases can be prepared.
Catt and coworkers have reported solid phase RIA techniques wherein the polymer of the solid phase is in form of powder (Biochem. J. 100:31 c (1966), plastic tubes (Science, 158:1570 (1967)) and plastic discs (J. Lab. and Clin. Med. 70:820 (1967)). In all cases the solid coated phase is maintained in a wet state and is freshly prepared or stored in a solution of bovine serum albumin. In U.S. Pat. No. 3,646,346, Catt discloses the use of an antibody coated test tube of polymeric material capable of adsorbing antibodies in RIA. The inside of the test tube is contacted with a buffered aqueous solution of the antibody, e.g. antiserum, for several hours, then the liquid is aspirated and the test tube is washed with saline. This coating procedure has serious disadvantages in that the resulting coating is not uniform, the attachment of the antibodies to the polymer surface is very poor, the concentration of antibody on the polymer surface is insufficiently low and inconsistent, and the coating is not sufficiently stable for routine laboratory handling.
Garrison et al (U.S. Pat. No. 3,790,663) discloses a similar method for binding antiserum to the surface of a plastic polymer, e.g., to the inside wells of a plastic microlitration tray. The wells are filled with antiserum which is diluted with a sodium borate buffer solution after one hour the antiserum is aspirated, the wells are rinsed and allowed to dry. The resulting coating has the same disadvantages as are mentioned above.
Crook et al. (U.S. Pat. No. 3,619,371) discloses a polymer matrix having biochemically active substances chemically bonded thereto. Into a polymer molecule triazinyl linking groups are chemically introduced and the triazinyl substituted polymer is then reacted with the biochemically active substance, e.g., an enzyme, antigen or antibody.